Supplementary Figure 1. Genotyping strategies for Mcm3 +/+, Mcm3 +/Lox and Mcm3 +/- mice and luciferase activity in Mcm3 +/Lox mice. A.

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Supplementary Figure 1. Genotyping strategies for Mcm3 +/+, Mcm3 +/Lox and Mcm3 +/- mice and luciferase activity in Mcm3 +/Lox mice. A. Upper part, three-primer PCR strategy at the Mcm3 locus yielding a 225 bp product in the wild-type allele and a 714 bp product in the Mcm3-Lox allele. Arrows indicate primer positions. Bottom part, genotyping examples of eight mice, of which #2 and #4 are Mcm3 +/Lox. B. Upper part, three-primer strategy yielding a 693 bp product in the wild-type Mcm3 allele and a 385 bp product in the Mcm3-null allele. Bottom part, genotyping examples of 10 mice, of which #1 to #7 were Mcm3 +/-. C. Top, bioluminescent signal in internal organs of Mcm3 +/Lox mice. Bottom, MCM3 protein level determined by immunoblot in extracts prepared from the same tissues. MEK2 levels are shown as reference. Full immunoblots are shown in Supplementary Fig. 9B. Tissue code: 1, pancreas; 2, small intestine; 3, testis; 4, spleen; 5, large intestine; 6, kidney; 7, liver; 8, seminal vesicle; 9, lung; 10, bladder; 11, stomach; 12, heart.

Supplementary Figure 2. Complete mrna sequencing of the Mcm3-Lox allele. A. Schematic representation of the Mcm3-Lox mrna. Left, PCR reactions confirming the presence of Mcm3-Lox mrna only in MEFs of Mcm3 +/Lox and Mcm3 Lox/Lox genotype. Right, PCR reactions showing amplification of three overlapping cdna fragments used for sequencing. B. Alignment of the experimental mrna-derived Mcm3-Lox sequence with the murine Mcm3 NCBI reference sequence (NM_008563.2) and the targeting vector used for mouse generation. Only the last four exons are shown for clarity. Numbers are positions from ATG (+1). The experimental sequence was identical

(100%) to that of the targeting vector. One silent point mutation (G-T) at position 2351 (highlighted in yellow) was found in both the experimental and vector sequences, relative to the reference sequence. The corresponding amino acid in MCM3 protein (Phe784) remained invariant. C. Alignment of the corresponding protein sequences. Numbers indicate positions from the +1 Met amino acid.

Supplementary Figure 3. DNA replication with reduced activation of dormant origins in Mcm3 Lox/Lox MEFs. A. BrdU incorporation profiles in Mcm3 +/+, Mcm3 +/Lox and Mcm3 Lox/Lox MEFs. The percentage of cells in the G1, S and G2/M phases of the cell cycle are indicated. B. Proliferation curves of Mcm3 +/+, Mcm3 +/Lox and Mcm3 Lox/Lox MEFs. Error bars represent SD of duplicates. C. Top left, outline of the experiment. MEFs were sequentially labeled with CldU (red) and IdU (green), in the absence or presence of 0.5 µm aphidicolin. Top right, schematic of a DNA molecule with two active origins, representing the fibers used to measure inter-origin distance (IOD). Bottom left, quantification of IOD of Mcm3 +/+, Mcm3 +/Lox and Mcm3 Lox/Lox MEFs. Median IOD values are indicated (70-100 IODs scored for each condition). Experiments were done in triplicate and statistical analysis was done with Fisher s test (** p<0.01; n.s. not significant). Bottom right, fiber examples. Scale bar, 10 μm. D. Confocal microscopy images of H2AX nuclear staining in Mcm3 +/+, Mcm3 +/Lox and Mcm3 Lox/Lox MEFs. Scale bar, 2.5 m. Right, quantification of H2AX nuclear intensity. Red lines indicate the median value (>700 nuclei scored per condition). Data are representative of 3 independent experiments. P-values were calculated by Mann-Whitney test (*** p<0.001; ns, not significant).

Supplementary Figure 4. Low expression of Mcm3 in the fetal liver of Mcm3 Lox/Lox embryos. A. Mcm3 mrna expression in the fetal liver of Mcm3 +/+, Mcm3 +/Lox and Mcm3 Lox/Lox embryos, as determined by qrt-pcr. Histogram bars represent average value SD of 3 independent experiments. B. Immunoblots showing total MCM3, H2AX and ps15-p53 protein levels in fetal liver extracts. For accurate comparisons, different amounts of each extract were loaded. In each SDS-PAGE, MEK2 levels are shown as loading control. Full immunoblots are shown in Supplementary Fig. 9C. C. Top, IHC detection of MCM3 protein in Mcm3 +/+, Mcm3 +/Lox and Mcm3 Lox/Lox wholeembryo (E16.5) sections. Scale bar, 5 mm. Lower panels, IHC of MCM3 and Ki67 and H2AX in the fetal liver. Scale bar, 25 M. Signal quantifications represents stainingpositive area (average value SD) in the fetal liver of Mcm3 +/+ (n=4 for MCM3, n=2 for Ki67); Mcm3 +/Lox (n=5 for MCM3, n=2 for Ki67) and Mcm3 Lox/Lox (n=5 for MCM3, n=2 for Ki67) embryos. P-values were calculated by Fisher s test (***p<0.001; n.s. not significant).

Supplementary Figure 5. IHC stainings of mature hematopoietic populations in fetal liver and thymus. IHC stainings of B-lymphocytes (Pax5), megakaryocytes (FVIII) and T-lymphocytes (CD3) in MCM3 +/+, MCM3 +/Lox and MCM3 Lox/Lox whole embryos. Scale bar, 5 mm. Magnifications show Pax5 and FVIII staining in the fetal liver and CD3 staining in the fetal thymus. Scale bar, 50 m.

Supplementary Figure 6. Detection of activated caspase 3 in Mcm3 Lox/Lox EBs. A. Examples of cytometric detection of activated caspase 3 (C3A) in R1-R5 EBs previously gated using CD71/Ter119. See Materials and Methods for details. B. Quantification of the percentage of C3A-positive cells in each population (n=5 Mcm3 +/+, n=6 Mcm3 +/Lox, n=6 Mcm3 Lox/Lox. * denotes p< 0.05 in One-way Anova test).

Supplementary Figure 7. Quantification of DNA fiber length in the experiments shown in Main Figure 5. Box-plot representation of DNA fiber lengths. A minimum of 200 fibers labeled with at least one replication structure (origin/fork/termination) were counted in each condition. The median value of fiber length is indicated in each case.

Supplementary Figure 8. Partial rescue of Mcm3 Lox/Lox embryonic lethality by Chk1 overexpression. A. Percentages of mice of the indicated genotypes (obtained/expected) derived from continued breeding of Mcm3 +/Lox : Chk1 +/Tg mice. B. Immunoblots showing MCM3 protein levels in whole fetal liver extracts of Mcm3 +/+, Mcm3 +/Lox, Mcm3 Lox/Lox embryos in the Chk1 +/Tg genetic background. The amount of MCM3 protein in Mcm3 Lox/Lox extracts is comparable to that of the C57BL/6 background, shown in Supplementary Fig. 4. Full immunoblots are shown in Supplementary Fig. 9D.

Supplementary Figure 9. Full immunoblots. This figure includes larger areas of the immunoblots corresponding to (A) Figure 1F; (B) Supplementary Figure 1C; (C) Supplementary Figure 4B; (D) Supplementary Figure 8B. Dashed boxes correspond to the area shown in the main figures.

Case Age (months) Diagnosis Location IHC BBG107 20 Lymphoma BBG115 30,5 Lymphoma Liver Lymphatic gland Kidney Adenomatous hyperplasia Thyroid BBG132 29 Follicular lymphoma Infiltration Lung Follicular adenoma Thyroid Epithelial hyperplasia Skin Polyp Intestine BBG185 28 Histiocytic sarcoma BBG193 27,5 Lymphoma Lymphoma infiltration Adipose tissue BBG602 18 Adenocarcinoma Harderian gland BBG621 18,5 Bronchioalveolar adenocarcinoma (II) Lung Hyperplasia Forestomach BBG656 19 Lymphoma Peripancreatic gland Supplementary Table 1. Summary of tumor incidence in MCM3 +/+ mice.

Case Age (months) Diagnosis Location IHC BBG51* 30 Adenocarcinoma Lung Hepatocarcinoma Liver Skin BBG52* 30 Histiocytic sarcoma Stomach Lung Lymphatic gland BBG54* 33 Histiocytic sarcoma Liver BBG55* 33 Histiocytic sarcoma Liver F4/80 Mesenteric glanglion Kidney BBG60* 28 Lymphoma PAX5/ CD3/ F4/80 BBG66* 22 Lymphoma BBG137* 23 Histiocytic sarcoma Liver CD3/ F4/80 Mesenteric glanglion BBG145* 26 Lymphoma Adipose tissue BBG150* 22 Adenocarcinoma Mammary gland BBG170* 28 Angiosarcoma Lymphatic glands Mesenteric glanglion BBG177* 23 Lymphoma Ureter Thymus Lung Bone marrow PAX5/ CD3 BBG181* 25 Lymphoma Liver Mesenteric ganglion Kidney BBG190* 25 Lymphoma Adenocarcinoma Lung BBG202* 21 Papilloma Stomach BBG203* 21 Lymphoma

Mesenteric ganglion Pancreas Lung Lymphoma Adipose tissue BBG235* 28 Mammary gland Hepatocarcinoma Liver Adenoma Small intestine Thyroid BBG261* 20 Lymphoma PAX5 Mesenteric ganglion PAX5/ CD3 BBG295* 20 Lymphoma Kidney Lung Mammary gland Lipoma Adipose tissue BBG335 23 Follicular hyperplasia Thyroid Bone CD31 BBG340* 20 Angiosarcoma Adipose tissue Lymphoma Adipose tissue Lymphatic glands Pancreas BBG427 21.5 Intestine Papilloma Stomach Adenomatous hyperplasia Thyroid Cystic hyperplasia Endometrium BBG518 23 Lymphoma Bone marrow Prostate Epididyme Lymphoma BBG632 21 Bone marrow Follicular hyperplasia Thyroid Adipose tissue BBG664 12 Leiomyosarcoma Skin Actin /p-p53

Histiocytic sarcoma Muscle F4/80 /CD3 BBG802 12 Adenoma Thyroid Lipoma Adipose tissue Supplementary Table 2. Summary of tumor incidence in MCM3 +/Lox and MCM3 +/Lox-Neo (*) mice.

Case Age (months) Diagnosis Location IHC Lymphatic glands Lymphoma Liver BBG111 30,5 Adrenal hyperplasia Kidney Papilloma Stomach Serous cystadenome Uterus Adenomatous hyperplasia Small intestine Peripancreatic gland CD3/ PAX5/ F4/80 Kidney Lymphoma Liver BBG264 21 Lung Folicular hyperplasia Thyroid gland Prostate hyperplasia Prostate BBG269 23 Adenocarcinoma Lung Lymphoma Pancreas CD3/ PAX5 BBG271 23 Follicular adenoma, mesenteric and thoracic CD3/ PAX5 ganglions Ductal hyperplasia Mammary gland BBG275 21 Adenocarcinoma Lung Hyperplasia Thyroid BBG277 23 Mesothelioma Mesenteric ganglion Hyperplasia Thymus BBG464 17,5 Lymphoma Peripancreatic gland BBG624 17 Hepatocarcinoma Liver Adenoma Small intestine Liver Adipose tissue BBG640 13 Histiocytic sarcoma Lung (B-cell type) Lymphatic ganglions Testis Bladder BBG642 18 Lymphoma Intestine

Liver Adrenal hyperplasia Kidney Hyperplasia Bladder Myeloid hyperplasia Bone Marrow Adenoma Thyroid/ Parathyroid BBG644 12 Hyperplasia (Leydig cells) Testis Pax5 BBG651 12 Lymphoma Lymphatic glands Adipose tissue Cholangioma Liver Cytokeratin 19/ CD31 BBG689 17,5 Lymphoma Kidney Follicular hyperplasia Thyroid BBG943 15,5 Hyperplasia Stomach Supplementary Table 3. Summary of tumor incidence in MCM3 +/- mice.

Allele Primer name Sequence (5 3 ) 1 GTGTCTGAGGGCATCGTCTT Mcm3-Lox 2 CCTCACAAGCCCAATCCTAA 3 GCAAGCTGACCCTGAAGTTC M TGCCTATGGCTCATCTGAAGAACTGC Mcm3-null 2 CCTCACAAGCCCAATCCTAA 4 TCTCCCAGTGTTCTTGAGGC Supplementary Table 4. Oligonucleotide primers used for mice genotyping. Gene Primer Sequence (5 3 ) Mcm3 MCM3-Fw TTCCTCAGCTGTGTGGTCTG MCM3-Rv TCACCACCCTAGTGGCTTTC Gapdh GAPDH-Fw TGAAGCAGGCATCTGAGGG GAPDH-Rv CGAAGGTGGAAGAGTGGGAG Supplementary Table 5. Oligonucleotide primers used for qrt-pcr. Gene Primer Sequence (5 3 ) Fw1 ATTTCGGAGCTGCGAC Fw3 CCATCACCATCCAGGAGATG Fw4 AAGCTGGTGCTATGGTCCTG Mcm3 Fw6 CGTTTCCGGACTGTTTGGT Rv2 CGATTCTTAAGTGATACTTGGTAGC Rv3 AGCAGAGGATTGCCTTCTTG Rv4 GAACTACCCAATGGCAAAGC Supplementary Table 6. Oligonucleotide primers for used for DNA sequencing.

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